PSMC3 promotes RNAi by maintaining AGO2 stability through USP14

Background Argonaute 2 (AGO2), the only protein with catalytic activity in the human Argonaute family, is considered as a key component of RNA interference (RNAi) pathway. Here we performed a yeast two-hybrid screen using the human Argonaute 2 PIWI domain as bait to screen for new AGO2-interacting proteins and explored the specific mechanism through a series of molecular biology and biochemistry experiments. Methods The yeast two-hybrid system was used to screen for AGO2-interacting proteins. Co-immunoprecipitation and immunofluorescence assays were used to further determine interactions and co-localization. Truncated plasmids were constructed to clarify the interaction domain. EGFP fluorescence assay was performed to determine the effect of PSMC3 on RNAi. Regulation of AGO2 protein expression and ubiquitination by PSMC3 and USP14 was examined by western blotting. RT-qPCR assays were applied to assess the level of AGO2 mRNA. Rescue assays were also performed. Results We identified PSMC3 (proteasome 26S subunit, ATPase, 3) as a novel AGO2 binding partner. Biochemical and bioinformatic analysis demonstrates that this interaction is performed in an RNA-independent manner and the N-terminal coiled-coil motif of PSMC3 is required. Depletion of PSMC3 impairs the activity of the targeted cleavage mediated by small RNAs. Further studies showed that depletion of PSMC3 decreased AGO2 protein amount, whereas PSMC3 overexpression increased the expression of AGO2 at a post-translational level. Cycloheximide treatment indicated that PSMC3 depletion resulted in a decrease in cytoplasmic AGO2 amount due to an increase in AGO2 protein turnover. The absence of PSMC3 promoted ubiquitination of AGO2, resulting in its degradation by the 26S proteasome. Mechanistically, PSMC3 assists in the interaction of AGO2 with the deubiquitylase USP14(ubiquitin specific peptidase 14) and facilitates USP14-mediated deubiquitination of AGO2. As a result, AGO2 is stabilized, which then promotes RNAi. Conclusion Our findings demonstrate that PSMC3 plays an essential role in regulating the stability of AGO2 and thus in maintaining effective RNAi. Supplementary Information The online version contains supplementary material available at 10.1186/s11658-022-00411-y.

. PSMC3 is required for mRNA cleavage rather than translational repression a. EGFP-expressing HeLa cells were transfected with control siRNA or siGFP. After 48 hours, digital images (lower panel) were obtained with a fluorescence microscope. Then cells were harvested and analyzed by Western blotting assays (upper panel). b and c. Depletion of PSMC3 abolishes the siRNAmediated cleavage of mRNA. A stable HeLa cell line expressing EGFP was transfected with the indicated siRNAs. After 48 hours, digital images (b) were obtained with a fluorescence microscope. Then cells were harvested and analyzed by Western blotting assays (c). d and e. PSMC3 is required for miR-21induced mRNA cleavage. A stable HeLa cell line expressing EGFP-miR-21 (which contains a sequence with 1 x perfect complementarity to miR-21 in its 3' UTR) was transfected with an miR-21 antisense oligomer or control oligomer as well as control siRNA or siRNAs against AGO2 or PSMC3. After 48 hours, digital images (d) were obtained with a fluorescence microscope. And whole-cell lysates were analyzed by Western blotting assays (e). f and g. Depletion of PSMC3 has no effect on translational repression. A stable HeLa cell line expressing EGFP-CXCR4 (which contains a sequence with 4 x bulged CXCR4 binding sites in its 3' UTR) was transfected with the indicated siRNAs. After 48 hours, digital images (g) were obtained with a fluorescence microscope. Then cells were harvested and analyzed by Western blotting assays (f). All results are representative of three independent experiments. Figure S3. Depletion of PSMC3 by siRNAs a. HeLa cells were transfected with control siRNA or AGO2 siRNAs (siAGO2#1, siAGO2#2 or siAGO2#3). Real-time RT-PCR analysis of AGO2 mRNA was performed using total RNA isolated from HeLa cells after 48 hours of transfection. In all statistical comparisons, three independent experiments were performed (mean ± S.D., n = 3, Student's t-test). ***, P < 0.001. b. HeLa cells were co-transfected with the indicated plasmids. Cell lysates were analyzed by Western blotting with anti-AGO2 and anti-GAPDH antibodies. c. HeLa cells were transfected with control siRNA or PSMC3 siRNAs (siPSMC3#1 or siPSMC3#2). Real-time RT-PCR analysis of PSMC3 mRNA was performed using total RNA isolated from HeLa cells after 48 hours of transfection. In all statistical comparisons, three independent experiments were performed (mean ± S.D., n = 3, Student's t-test). **, P < 0.01, ***, P < 0.001. d. The region of PSMC3 targeted by siPSMC3#2 is indicated. Three-point mutations were introduced into the siRNA-binding site without affecting the PSMC3 amino acid sequence. subjected to immunofluorescent staining with antibody against AGO2. Cells were stained with DAPI to visualize nuclei, and the images were digitally merged. Scale bar, 10 μm. b. AGO2 has no effect on PSMC3 protein levels. HeLa cells were co-transfected with the indicated plasmids. Then nucleoplasmic or cytoplasmic extracts were harvested and analyzed by Western blotting assays. All results are representative of three independent experiments. Figure S5. Determination of the AGO2 ubiquitination sites a. The putative ubiquitination sites in the AGO2 C-terminus were predicted by BDM-PUB and UbPred.
b. Schematic representation of AGO2 mutants with C-terminal K-to-R (lysine residues replaced by arginines) changes. c. Multiple C-terminal lysine residues target AGO2 for ubiquitination. HeLa cells were transfected with HA-tagged wild-type (wt) AGO2 or AGO2 with C-terminal K-to-R mutations.
After 36 hours, cells were split into two aliquots; one was treated with 30 μM MG132 and one with DMSO for 10 h. Total cell extracts were immunoprecipitated with an anti-HA antibody and probed in   h. The EGFP value was normalized by the RFP value. Data are presented as the mean ± standard deviation for three independent experiments. **, P < 0.01.

Figure S8. USP14 protein is localized in cytoplasm
Western blotting assays were used to detect USP14 protein levels in nucleoplasm or cytoplasm from HeLa cells.

Plasmids and oligos
The coding region of the human AGO2 PIWI domain was cloned in frame with the hSOS domain of pSOS (Stratagene, Santa Clara, CA). To generate HA-tagged AGO2 PIWI, the fragment of the human AGO2 PIWI domain was inserted into a modified pcDNA3 plasmid (Invitrogen, Carlsbad, CA) containing an N-terminal 3 x HA-tag. Full-length AGO2 was amplified by PCR using the primers 5'-GCC ACC GAA TTC GCC ACC ATG TAC TCG GGA GCC-3' and 5'-GCC ACC ATG CGT CGA CGC AGC AAA GTA CAT GGT GCG CAG-3' and cloned into the EcoRI and SalI restriction sites of pcDNA3 with a 3 x HA-tag or a 3 x Flag-tag. Multiple K-to-R mutants in AGO2 were generated by site-directed mutagenesis of HA-AGO2 using a PCR-based strategy. To obtain HA-, Myc-and Flag-tagged PSMC3, The EGFP gene from the pEGFP-N2 vector (Clontech, Mountain View, CA) was subcloned into pcDNA3 and is referred to as pcDNA3/EGFP. Reporter plasmid pcDNA3/EGFP-miR-21 (1 × perfect) was generated by PCR as described previously 44 . The EGFP reporter construct carrying four bulged binding sites for CXCR4 siRNA antisense was subcloned from the pRL-TK CXCR4 4 × plasmid (Addgene, Cambridge, MA) into pcDNA3/EGFP 3' UTR using the XbaI and ApaI sites. All constructs were checked by sequencing and BLAST searching. Yeast two-hybrid screen A Stratagene Cytotrap system human lung library (La Jolla, CA, US) was screened according to the manufacturer's instructions. The pSOS bait construct (pSOS/AGO2 PIWI) and pMyr target library were co-transformed into S. cerevisiae strain cdc25H and plated on synthetic glucose minimal medium lacking leucine and uracil. After 2-4 days at 25°C, replica plates of the transformants were made and transferred to a 37°C incubator for 6 days. Putative positive clones were picked, and yeast plasmid DNA was isolated and transformed along with the pSOS bait construct into yeast for another interaction test. Positive clones were subsequently sequenced, and the identification of each clone was determined by performing BLAST searches against the human genome.

Cell culture and transfection
HeLa cells were inoculated in cell culture plates or cell culture flasks and transfected when the cell density was 50%-80%. Lipofectamine 2000 and plasmid or siRNA were diluted with appropriate amounts of Opti-MEM, respectively. After 5 min, they were mixed and incubated for 20 min. Then the mixture was added to the culture plate or culture flask and replaced with the appropriate volume of cell culture medium after 6 h. The amount of siRNA was 100 nM, and the amount of plasmid was 1μg/ml. The corresponding assays were performed 48 hours after transfection.

Co-immunoprecipitation and immunofluorescence
At 48 h post-transfection, HeLa cells were lysed with buffer containing 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM NaF, 0.1% NP-40, 10% glycerol and complete protease inhibitor cocktail (Roche) at 4ºC for 30 min. After centrifugation, 1 ml of the resulting supernatant was incubated with anti-AGO2, anti-HA or anti-PSMC3 antibodies at 4°C for 16 h. The protein-antibody complexes were precipitated by incubating the lysate with Protein A/G UltraLink Resin (Pierce, IL, USA) for 5 h under constant rotation at 4°C. After incubation, the resin was washed four times with lysis buffer, and proteins were eluted with 2×loading buffer, boiled for 5 min, and processed for Western blotting.
For immunofluorescence detection, transfected HeLa cells that had been grown on slides were fixed using 4% paraformaldehyde in 1×PBS for 30 min at 4°C. Permeabilization was performed with 0.2% Triton-X 100 for 5 min at 4°C. Samples were blocked for 30 min in 1×PBS containing 10% (w/v) donkey serum. All primary and secondary antibodies were diluted in 2% donkey serum. Samples were incubated with the anti-AGO2 primary antibody (1:500 dilution), anti-PSMC3 primary antibody (1:500 dilution), anti-Myc primary antibody (1:1000 dilution), anti-HA primary antibody (1:500 dilution) or anti-Flag primary antibody (1:1000 dilution) in a humid chamber overnight at 4°C. After washing with 1×PBS, the slides were incubated with the TRITC/FITC-conjugated secondary antibody (1:200 dilution) for 1 h at 4°C. Finally, the cells were stained with 0.05 g/ml DAPI in PBS for 2 min at RT. Mounted slides were viewed in a confocal microscope, and image analysis was performed using Leica Confocal Software and Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA).

Preparation of cytoplasmic and nuclear fractions
The subfractionation of transfected cells into nuclear and cytoplasmic extracts was performed as previously described. Briefly, at 48 h post-transfection, 2×10 6 cells were collected by trypsin/EDTA